1. Field
This disclosure is concerned generally with a novel cytokine antagonist preparation and specifically with the preparation, characterization, and use of an Interleukin-6 inhibitor which can be isolated from tissue culture fluid and has been found to have in vitro Interleukin-6 antagonist activity.
2. Background
The involvement of Interleukin-6 (IL-6) in human health and disease is under intensive investigation. Elevated levels of IL-6 have been found in the bloodstream and/or body fluids of individuals with bacterial and viral infections, trauma, autoimmune disorders, and neoplasias. Correlations of IL-6 levels with severity of symptoms and the beneficial effect of anti-IL-6 antibodies in animal models suggest that the cytokine may play a pathophysiological role in some disease indications. Antagonists of IL-6 may therefore be of therapeutic use.
A specific, natural IL-6 antagonist has yet to be described. Portier et al. (Blood, 81(11):3076-82 (1993)) found that .gamma.-interferon (.gamma.-IFN) will inhibit IL-6 dependent myeloma cell growth but .gamma.-IFN does not inhibit IL-6 activity in other types of in vitro assays.
Brakenhoff et al. (J. Biol. Chem., 269(1):86-93 (1994)) engineered biologically inactive IL-6 mutants which bound to the 80 kD IL-6R but did not bind to gp130, thus preventing signal transduction. These mutant proteins acted as IL-6 antagonists by preventing native IL-6 from binding to the IL-6 receptor subunits. However, the mutant protein's potential immunogenicity could be a difficulty for therapeutic use.
Klein et al. (Blood, 78:1198-1204 (1991)) found that administration of a murine anti-IL-6 antibody to a patient with leukemia blocked myeloma cell proliferation in the bone marrow. Again though, because the murine antibody is a foreign protein, there is the potential for immunogenicity.
It has been postulated that bioengineered derivatives of a soluble 80 kD receptor would act as an IL-6 antagonist by binding to circulating IL-6 but not to gp130 thus preventing signal transduction (J. Bauer, Biotechnology Therapeutics, 2(3&4):285-298 (1991)). However, these proteins might have an epitope that could be recognized as foreign and could still be immunogenic if used as a therapeutic. Bauer also stated that clinical trials using human anti-human IL-6 antibodies for the treatment of multiple myelomas have begun (Id.). At this time, the outcome of the clinical trials is unknown.
Monocytes/macrophages have been shown to produce both cytokines and cytokine inhibitors, such as the IL-1 inhibitor Roberts et al. found in Respiratory Syncytial Virus (RSV) infected monocytes (J. Exp. Med., 163:511-519 (1986)) and the IL-1 receptor antagonist protein (Janson et al., J. Immunol., 147(12):4218-4223 (1991)). In this invention, we investigated the possibility that such cells may also secrete an IL-6 inhibitor. Since it was difficult to establish a consistent supply of human peripheral blood monocytes, we utilized the human promyelocytic leukemia cell line, HL-60. Treatment of HL-60 with phorbol diesters induces differentiation to cells exhibiting several characteristics of macrophages (Hall et al., Cell. Immunol., 76:58-68 (1983)), while dimethyl sulfoxide (DMSO) or retinoic acid (RA) treatment results in differentiation along the granulocytic pathway (Leftwich et al., Canc. Res., 46:3789-3792)). We found that exposure of HL-60 cells to phorbol diesters specifically induced secretion of an IL-6 inhibitor. It appears that this IL-6 inhibitor is an apparently novel human protein. Because the HL-60 cell line is human, the IL-6 inhibitor should contain the human amino acid sequence and therefore not be immunogenic in vivo. This would be an improvement over the prior examples of IL-6 antagonists.